Two days ago I posted on the development of DNA scaffolding with programmable modules for use in the modular molecular composite nanosystems (MMCNs) route to atomically precise productive nanosystems. Two months ago I posted a story about the incorporation of DNA devices in a DNA scaffolding for the purpose of manipulating molecular building blocks. Two recent publications in Nature Nanotechnology provide more evidence of the growing capability of DNA scaffolds to support complex and interactive functions.

In the first paper (abstract), scientists at Arizona State University were able to precisely position two molecules that individually bound to different portions of the protein molecule thrombin and show that the thrombin was bound to the DNA scaffold when the two binding molecules were attached to the DNA scaffold at a distance approximating the distance between the two binding sites on the thrombin molecule. From the abstract:

Using atomic force microscopy, we demonstrate direct visualization of high-affinity bivalent ligands being used as pincers to capture and display protein molecules on a nanoarray. These results illustrate the potential of using designer DNA nanoscaffolds to engineer more complex and interactive biomolecular networks.

Rectangular DNA tiles were assembled by the scaffolded DNA origami method such that two different single strand DNA sequences (aptamers), each of which binds to the blood coagulation protein thrombin, would be separated on the DNA tiles by a predetermined distance. The two aptamer sequences were chosen because they were known to bind to sites almost on opposite sides of the thrombin molecule (about 4.1 nm apart). The researchers hypothesis was that by systematically varying the distance between the aptamer sequences on the DA origami tile, an optimum distance would be found such that the two aptamers would act as one bivalent binding species, binding the thrombin protein much more tightly than would occur with either aptamer alone. The researchers’ expectations were met in that no binding was detected with either aptamer alone, only very little with the two aptamers separated by 2 nm, moderate binding with a 3.5 nm separation, better binding with a 5.3 nm separation, and decreased binding when the separation was too great—6.9 nm. These results justify the researchers optimism for using DNA scaffolds to create complex functional interactions:

This study also represents the first example of using the spatial addressability of self-assembled DNA nanoscaffolds to control multi-component biomolecular interactions and to visualize such interactions at a single-molecule level. … It may also be possible to use addressable DNA nanoscaffolds… to position motor proteins at a particular inter-molecular distance to display complex motor behaviours on a well-defined nanoscale landscape, generated, for example, by modifying the staple strands of the origami at different locations.

In a second paper (abstract), scientists from The Hebrew University of Jerusalem demonstrated the use of DNA scaffolds to organize enzymes and cofactor molecules so that enzymatic reactions can occur that will not occur when the same components are simply mixed in solution. From the abstract:

Here, we report the self-assembly of a DNA scaffold made of DNA strips that include ‘hinges’ to which biomolecules can be tethered. We attach either two enzymes or a cofactor-enzyme pair to the scaffold, and show that enzyme cascades or cofactor-mediated biocatalysis can proceed effectively; similar processes are not observed in diffusion-controlled homogeneous mixtures of the same components. Furthermore, because the relative position of the two enzymes or the cofactor-enzyme pair is determined by the topology of the DNA scaffold, it is possible to control the reactivity of the system through the design of the individual DNA strips. This method could lead to the self-organization of complex multi-enzyme cascades.

In this case the DNA scaffolds used were ladders that were either two or four DNA units wide. Each DNA unit was a single-strand ‘hexagon-like’ structure composed of a loop of 60 bases with an attached strand of 10 bases to act as a tether to attach other molecules to the unit. Due to complementary sequence relationships designed into the units, two types of units could hybridize to form long strips or ladders two units wide with a unique tether on each of the two strips of units, and four types of somewhat longer units could hybridize to form long ladders four units wide but with (unique) tethers only on the first and fourth strip of units (the two outside strips). The two-unit-wide ladders were found by AFM to be 13 nm wide and the four-unit-wide ladders to be 33 nm wide. The enzyme horseradish peroxidase was attached to a single strand of DNA that hybridized to one type of tether and the enzyme glucose oxidase was attached to a DNA strand that hybridized to the other type of tether. The result using the two-unit-wide ladders was a strip of horseradish peroxidase molecules separated by 13 nm from a row of glucose oxidase molecules. With the four-unit-wide ladders, the two rows of enzyme molecules were separated by 33 nm. The product of the reaction catalyzed by glucose oxidase acts as a substrate for horseradish peroxidase. This reaction cascade is not observed in the absence of the DNA scaffold but was observed when the enzymes were attached to the DNA scaffolds. The two-unit-wide ladder worked better than the four-unit-wide ladder because the two different enzymes were held closer together to facilitate the product of one reaction becoming a substrate for the other.

A similar demonstration of the power of using scaffolds to organize molecules was obtained in a separate experiment in which the two-unit-wide DNA scaffold were used to spatially arrange a third enzyme and a cofactor molecule necessary for enzyme activity, but with tethers of varying length used to vary the distance between enzyme and cofactor. No activity was seen in the absence of a scaffold to hold the cofactor near the enzyme. In this case a longer tether worked better because the shorter tether did not allow contact between the enzyme and the cofactor.

A News and Views commentary in the same issue praises the research as “an excellent example of programmable DNA-directed assembly of multi-component enzymatic complexes” that “is modular and could therefore be adapted for the construction of other functional molecular complexes”, but notes that truly functional systems will require expanding from two-dimensional DNA-directed assembly to three-dimensional DNA scaffold motifs, and incorporating DNA nanomechanical devices to achieve dynamic complexes.

Indeed, it is foreseeable that, by merging the addressability of DNA structure with the versatile functionality of protein libraries, self-assembling, dynamic and functional biomolecular networks are in reach.

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